Project Summary/Abstract Alphaviruses are small, enveloped plus strand RNA viruses and include serious human pathogens such as the encephalitis viruses and Chikungunya virus. Despite the risk they pose to human health, we lack a clear understanding of how alphaviruses assemble into viral particles, a stage in the viral lifecycle that is ill-defined for most enveloped viruses. Specifically, the mechanism of nucleocapsid (NC) assembly and incorporation into alphaviral particles is largely unknown. The NC is made up of a spherical lattice of capsid protein (Cp) surrounding the viral RNA genome (gRNA), and its assembly is required for the production of infectious virus. Determining the mechanism of NC assembly has been difficult due to the presence of several distinct Cp/NC pools that form during an infection. Only a fraction of total Cp produced during an infection is incorporated into progeny virus and it is unclear which pool/s is used and how this occurs. In this project, I will investigate the pathway of Cp assembly into alphavirus NC by identifying which Cp/NC pools are incorporated into the virus, defining how Cp is transported to virus budding sites at the plasma membrane, and determining how Cp selectively packages gRNA during NC assembly. To do this, I have established a biotinylation system to track Cp during an infection using Streptavidin in biochemical and imaging techniques. I inserted the optimized biotin acceptor peptide (AVI tag) at a permissive site on Cp (Cp-AVI) and showed that the AVI tag does not perturb Cp?s biology. I have created cell lines that stably express the BirA biotin ligase at specific subcellular locations and shown that biotinylation of Cp-AVI by BirA is robustly detectable in microscopy and western blot studies. I have initiated biotin pulse-chase experiments where targeted BirA cell lines are pulsed with biotin and chased in biotin-free media to induce biotinylation of specific Cp-AVI pools at defined times post-infection. I will track Cp-AVI pools by assessing their localization and transport over time, and determine which Cp pools contribute to mature virus by scoring for biotinylated Cp-AVI in released viral particles. I will determine if Cp-AVI transport to virus budding sites occurs through an active transport mechanism, possibly by co-trafficking with the viral E2 transmembrane glycoprotein, by testing how vesicular transport inhibitors affect Cp-AVI localization and by correlating Cp-E2 binding with their co-localization to virus budding sites over time. Lastly, I will determine how Cp specifically packages gRNA by using PAR-CLIP (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation) time course experiments to define the contacts Cp makes with the gRNA during NC assembly. I will identify gRNA sites Cp specifically recognizes and compare Cp-gRNA contacts to those in the released viral particle. The results from this project will provide critical insights on the molecular mechanism of alphavirus NC assembly and improve our fundamental understanding of the exit of enveloped viruses.